The overall goals of this proposal are to understand how the transmembrane domain (TMD) of Pmel17 regulates its sorting to melanosomes and its ability to form the fibrillar melanosome matrix, and how mutations in this domain result in the melanosome dysfunction underlying pigmentation defects. Melanosomes are pigment-cell specific organelles in which melanins, which protect the skin and eyes from UV radiation, are synthesized and stored. Within melanosomes, melanins are deposited on a fibrillar amyloid-like matrix of which the highly conserved pigment cell-specific protein Pmel17 is the main component. To form fibrils, Pmel17 - like several pathological amyloid precursors - must first be sorted within endosomal intermediates to intralumenal vesicles (ILVs), but the mechanisms responsible for this sorting are unclear. Natural occurring mutations in the unusually long Pmel17 TMD cause hypopigmentation in model organisms; although similar mutations in human Pmel17 have not been identified in pigmentary diseases, they likely exist given the high degree of homology among vertebrate Pmel17 orthologues and the similar phenotypes associated with Pmel17 mutations in a broad range of species. The hypopigmentation phenotypes associated with these mutations suggest that the TMD is critical to Pmel17 function. This proposal poses the hypothesis that the Pmel17 TMD mediates partitioning into lipid rafts and that this partitioning is required for sorting of Pmel17 to ILVs for subsequent fibril formation because long TMDs tend to partition in lipid rafts and ILVs are enriched in lipid raft components. This hypothesis will be addressed with the following specific aims: [unreadable] [unreadable] 1) Determine if Pmel17 associates to lipid rafts using classical biochemical fractionation and test whether cholesterol depletion affects Pmel17 lipid raft association, sorting, and/or fibril formation. [unreadable] [unreadable] 2) Determine if mutations in the Pmel17 TMD affects sorting to ILVs and/or fibrillogenesis. [unreadable] [unreadable] The proposed work is relevant as it relates to understanding the mechanisms that control the targeting of Pmel17 to melanosomes and the formation of the amyloid-like matrix, essential to PrneM 7 function and melanosome integrity. The results may also be relevant to understanding how amyloid forms in neurodegenerative diseases. [unreadable] [unreadable] [unreadable]